Project Summary/Abstract RNA interference (RNAi) was originally described as a gene silencing mechanism triggered by the experimental introduction of double stranded (ds)RNA into the nematode C. elegans (Fire et al., 1998). The term RNAi is now used to refer to a diverse set of gene regulatory mechanisms that share common features including the involvement of a short 21-30 nucleotide (nt) long RNA and a protein cofactor of the Argonaute (RNase H-related) protein family. As an experimental tool, RNAi is of broad relevance to basic medical research in numerous fields, and RNAi therapeutics are now under development for several clinical applications. Furthermore, RNAi-related mechanisms function in conserved gene-regulatory pathways that are of basic and fundamental importance to human cellular and developmental biology. The proposed genetic and biochemical studies will advance our understanding of RNAi and related pathways. The ability to combine classical genetics with the newer disciplines of deep-sequencing, functional genomics and proteomics, make C. elegans an ideal system for these studies. In all animals studied to date, multiple RNAi-related pathways co-exist within cells. In C. elegans, three AGO pathways have the potential to mediate genome-wide or transcriptome-wide surveillance. These pathways are: (i) the WAGO pathway, which targets transposons, pseudogenes and other cryptic loci, as well as some protein encoding genes; (ii) the CSR-1 pathway, which targets most, if not all, protein-encoding mRNAs expressed in the germ line; and (iii) the PRG-1 pathway, which targets at least one transposon family but has more than fifteen thousand additional genomically-encoded small RNA cofactors whose targets are not known. An important goal of this work is to understand how these distinct pathways identify targets and mediate specific regulatory outcomes. The proposed studies will investigate the function and interrelationship of these pathways, using an array of biochemical, molecular and genetic approaches. The mechanisms and protein families that mediate RNAi are highly conserved in animals, therefore, insights from the proposed studies will be directly relevant to human biology and disease. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page